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Amplite® Colorimetric Calcium Quantitation Kit *Blue Color*

Calcium dose response was measured on a 96-well black wall/clear bottom plate with the Amplite® Colorimetric Calcium Quantitation Kit. As low as ~ 2.5 µM Ca2+ was detected with 5 minutes incubation time (n=3)
Calcium dose response was measured on a 96-well black wall/clear bottom plate with the Amplite® Colorimetric Calcium Quantitation Kit. As low as ~ 2.5 µM Ca2+ was detected with 5 minutes incubation time (n=3)
Calcium dose response was measured on a 96-well black wall/clear bottom plate with the Amplite® Colorimetric Calcium Quantitation Kit. As low as ~ 2.5 µM Ca2+ was detected with 5 minutes incubation time (n=3)
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H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22


Calcium is essential for all living organisms, particularly in cell physiology, where movement of the calcium ion Ca2+ into and out of the cytoplasm functions as a signal for many cellular processes. Calcium is the fifth most abundant element by mass in the human body, where it is a common cellular ionic messenger with many functions, and serves also as a structural element in bone. Calcium plays an important role in mediating the constriction and relaxation of blood vessels, nerve impulse transmission, muscle contraction, and hormone secretion. The serum level of calcium is closely regulated within a fairly limited range (9 to 10.5 mg/dL) in the human body. Both hypocalcaemia and hypercal caemia are serious medical disorders. Causes of low calcium levels include chronic kidney failure, vitamin D deficiency, and low blood magnesium levels that can occur in severe alcoholism. Amplite® Calcium Detection Kit provides a simple method for detecting calcium in physiology solutions. This kit uses our Calcium Blue™ as the chromogenic calcium indicator. Its absorbance changes in response to calcium binding. Calcium Blue™ binds calcium tightly in the neutral pH range, generating Calcium Blue™-calcium complex that has intense absorption at ~650 nm.


Absorbance microplate reader

Absorbance600 or 650 nm
Recommended plateClear bottom


Example protocol


Protocol Summary
  1. Prepare test samples and calcium standard solution (50 µL)
  2. Add Calcium Blue™ reagent (50 µL)
  3. Incubate at room temperature for 5-10 minutes
  4. Monitor absorbance intensity at 600 or 650 nm 
Important      Thaw all the kit components at room temperature before starting the experiment.


Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

Calcium standard solution (3 mM)
Add 10 µL of Calcium Standard (300 mM) (Component C) to 990 µL Dilution Buffer (Component B) to get Calcium standard solution (3 mM) and mix well.


For convenience, use the Serial Dilution Planner:

Calcium standard


Table 1. Layout of calcium standards and test samples in a clear bottom 96-well microplate. CS = Calcium standard (CS1-CS7); BL = blank control; TS = test sample.
Table 2. Reagent composition for each well
CS1 - CS750 µLSerial Dilutions
BL50 µLDilution Buffer (Component B)
TS50 µLtest sample
Table 3.
Calcium StandardBlank ControlSerum or Urine
Serial Dilutions: 50 µLDilution Buffer (Compound B): 50 µL50 µL

Calcium assay
  1. Add the serially diluted calcium standards from 150 µM to 2.34 µM into wells from CS1 to CS7 in duplicate.
  2. Add 50 µL of Calcium Blue™ (Component A) to each well of calcium standards, blank control, and test samples to make the total calcium assay volume to 100 µL/well.
    Note     For a 384-well plate, add 25 µL of sample and 25 µL of assay reaction mixture into each well.
  3. Incubate the reaction for 5 to 10 minutes at room temperature, protected from light.
  4. Monitor the absorbance intensity with an absorbance plate reader at OD 600 nm or 650 nm. 

Assay Protocol for Serum and Urine Samples
  1. Take 10 μL of 300 mM Calcium Standard solution (Component C) to 990 µL Dilution Buffer (Component B) to get 3 mM Calcium Standard Solution.
  2. Take 500 μL of 3 mM Calcium Standard Solution to perform 1:2 serial dilutions to get 1.5, 0.75, 0.375, 0.1875, 0.094, 0.047 and 0 mM serially diluted Calcium standards.
  3. Add 10 μL of calcium standard, serum or urine samples and blank control into their respective wells.
  4. Add 200 μL of Calcium Blue™ (Component A) to each well of calcium standard, blank control, and test samples to make the total calcium assay volume of 210 µL/well.
    Note     For a 384-well plate, add 2.5 μL of sample and 50 μL of assay reaction mixture into each well.
  5. Incubate the reactions for 5-10 minutes at room temperature (protected from light).
  6. Measure the absorbance intensities at 600 nm or 650 nm. 



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Journal: (2019)
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